Abstract Understanding embryonic development requires knowledge of all the molecules produced as the zygote differentiates into the three primary germ layers of the embryo. Four decades of innovative embryological manipulations, testing of gene functions one gene at a time, and recently, Next-Generation Sequencing have identified multiple transcripts and abundant proteins that are essential to the patterning of the vertebrate embryo. However, very little is known about the total array of proteins and their post-translational modifications that contribute to the formation of the germ layers, and next to nothing is known about the contribution of small molecules (called metabolites) to these processes. To date, systems biology has defined the spatial and temporal changes of mRNAs, abundant proteins, and metabolites in the whole embryo, but it has been technologically impossible to utilize high-resolution mass spectrometry (HRMS), the gold standard technology for small molecules, to study hundreds-to-thousands of metabolites and proteins in single embryonic cells in the vertebrate embryo. The proposed research program fills this enormous knowledge and technological gap by utilizing novel single-cell mass spectrometry technologies to understand cell molecular processes that contribute to the formation of the three germ layers required for the successful patterning of the vertebrate frog (Xenopus laevis) embryo, a favorite model in cell/developmental biology. Most recently, single-cell mass spectrometry discovered metabolites capable of altering the normal cell fates of embryonic cells, suggesting that the complete molecular players are not yet fully identified or understood for germ layer induction. The proposed research program will determine this missing link in the understanding of molecular mechanisms governing vertebrate development. This work will integrate quantitative single-cell mass spectrometry, cell fate tracking, and gene knock-down experiments to determine how a targeted set of small-molecular reactions impact the formation of signaling centers required for dorsal axis specification. The outcomes of this interdisciplinary approach will help illuminate the role of the proteome and metabolome for the establishment of these important precursors. Because these molecular processes are highly conserved across vertebrates, the data collected from Xenopus are likely to have high relevance to human structural birth defects. The new biochemical information that will be obtained in individual embryonic cells and their progeny (cell lineage) at several critical developmental time points will also advance other research fields that involve cell differentiation (e.g., of stem cells) and the developmental origins of adult disease.